1. Field of the Invention
The present invention relates to the field of producing biomolecules, in particular polynucleotides like plasmid DNA. In particular, the present invention relates to a method on a manufacturing scale that includes cell lysis under alkaline conditions followed by neutralization and subsequent clarification of the cell lysate.
2. Related Art
The advances in molecular and cell biology in the last quarter of the 20th century have led to new technologies for the production of biomolecules (biopolymers). This group of naturally occurring macromolecules includes proteins, nucleic acids and polysaccharides. They are increasingly used in human health care, in the areas of diagnostics, prevention and treatment of diseases.
Recently some of the most revolutionary advances have been made with polynucleotides in the field of diagnostics, gene therapy and nucleic acid vaccines. Common to these applications is the introduction of DNA or RNA into cells with the aim of a diagnostic, therapeutic or prophylactic effect.
Polynucleotides are a heterogeneous group of molecules in terms of size, shape and biological function. Common to all of them are their building blocks (nucleotides as Adenine (A), Guanine (G), Cytosine (C), Thymine (T), Uracil (U)) and their high negative charge under physiological conditions. Representative members of polynucleotides are RNA (messenger RNA, transfer RNA, ribosomal RNA), genomic DNA (gDNA) or chromosomal DNA (cDNA), and plasmid DNA (pDNA). These macromolecules can be single- or double-stranded. Similar to proteins, they are able to build three-dimensional structures and aggregates under distinct conditions. Polynucleotides are sensitive to enzymatic degradation (DNases and RNases) and shear forces, depending on their size and shape. Especially chromosomal DNA, in its denatured and entangled form, is highly sensitive to mechanical stress, resulting in fragments with similar properties to pDNA. This becomes more and more critical with the duration of the shear force exposure (Ciccolini L A S, Shamlou P A, Titchener-Hooker N, Ward J M, Dunnill P (1998) Biotechnol Bioeng 60:768; Ciccolini L A S, Shamlou P A, Titchener-Hooker N (2002) Biotechnol Bioeng 77:796).
Plasmids (pDNA) are double stranded extrachromosomal circular polynucleotides. A typical plasmid contains between 1 and 20 kilo base pairs which corresponds to 3×106-13×106 Da and several thousand Å. Different topological forms of pDNA can be distinguished. The supercoiled (sc) or covalently closed circular (ccc) form is considered as most stable for therapeutic application and is therefore the desired form. The other topological pDNA forms are derived from the ccc form by either single strand nick (open circular or oc) or double strand nick (linear). Breakage of the strands can be caused by physical, chemical or enzymatic activity. For therapeutic use the percentage of ccc form is the main-parameter for assessing the quality of the pDNA preparation.
Therapeutic treatment based on pDNA is considered to be an alternative to treatment with classical chemical drugs or recombinant proteins. Due to the increasing amounts of pDNA required for preclinical and clinical trials, there is a demand for processes that can be performed on a manufacturing scale. These production processes must fulfill regulatory requirements (FDA, EMEA) and should be economically feasible.
In the past, the majority of biotechnological production processes have been developed for manufacturing of purified recombinant proteins. Due to the differences in the physico-chemical properties between polynucleotides and proteins, these methods cannot easily be adapted for the production of polynucleotides. Thus, there is a need for methods that are applicable to polynucleotides, in particular for production of plasmid DNA on a manufacturing scale.
In brief, a process for producing recombinant biomolecules, which are not secreted by the host, in particular DNA and large proteins, follows the steps of:                a) Fermentation (cultivation of cells that carry the biomolecule of interest and optionally harvesting the cells from the fermentation broth),        b) Disintegration of the cells (release of the biomolecule of interest from the cells),        c) Isolation and purification (separation of the biomolecule of interest from impurities).        
These steps are more specifically characterized for the production of polynucleotides, in particular for the production of pDNA, as follows:
Currently, E. coli is the most commonly used host for pDNA production. Other bacterial, yeasts, mammalian and insect cells may also be used as host cells in the fermentation step. Selection of a suitable host strain is of major importance for the pDNA quality. A high cell density and plasmid copy number and its stable maintenance during the fermentation are crucial for a robust economic process. For this purpose, a well-defined culture medium is needed. The end point of fermentation and the conditions during cell harvest, which usually follows fermentation, contribute to the quality of the polynucleotide (Werner R G, Urthaler J, Kollmann F, Huber H, Necina R, Konopitzky K (2002) Contract Services Europe, a supplement to Pharm. Technol. Eur. p. 34).
After fermentation, the cells are usually harvested, mostly by means of centrifugation. The harvested wet biomass is resuspended in an appropriate buffer. Before final isolation (by e.g. column chromatography, ultradiafiltration, extraction or precipitation) of the polynucleotide of interest from proteins, gDNA, RNA and other host related impurities, the cells need to be processed, either directly or after freezing and thawing. Alternatively to harvesting and resuspending the cells before further processing, the fermentation broth per se may be subject to further processing (WO 97/29190).
Processing starts with disintegration of the cells and ends with the first isolation step of the polynucleotide of interest, which is also termed “capture step”.
Disintegration of the cells can be achieved by physical, chemical or enzymatic methods. Most of currently available procedures were developed for the release of proteins from the cells and can not be used for polynucleotides without certain adaptations. Limitations of the established techniques are due to the differences of the physico-chemical properties between proteins and polynucleotides. High-pressure homogenization, the most common technology for the recovery of proteins, cannot be used for polynucleotides due to their size-depending shear force sensitivity and possible destruction of gDNA. (Carlson A, Signs M, Liermann L, et al. (1995) Biotechnol Bioeng 48:303). Chemical (Foster D (1992) Biotechn 10, (12):1539) and enzymatic (Asenjo J A, Andrews B A (1990) Bioprocess Technol 9: 143) methods cause minimal mechanical stress and minimal irreversible deterioration of the plasmid. Since it is the gentlest method, enzymatic disintegration utilizing lysozyme is the method of choice on laboratory scale. Typically, lysozyme is animal-derived (most commonly from chicken egg white) and therefore its use is a potential health risk (prions) and is considered as problematic by regulatory authorities like FDA or EMEA. Using recombinant lysozyme involves high raw material costs and analytical efforts. Thermal treatment of the cells is another option for a disintegration technique that avoids shear forces, as described in WO 02/057446 A2 and WO 96/36706. The suspension of microorganisms processed by short time exposure (30 seconds to some minutes) to 80° C. in a sink heater or in a filter (with filtering aids). This method is usually carried out in combination with a detergent (e.g. TRITON®) and lysozyme.
Usually, disintegration and release of plasmid DNA from bacterial cells is performed by alkaline lysis (a chemical method) as described by Birnboim and Doly (Birnboim H C, Doly J (1979) Nucl Acids Res 7: 1513).
The disintegration/release process disclosed therein can be divided into two steps, the first one being the intrinsic cell disintegration or lysis step and the second one being the neutralization step.
During alkaline lysis, cells are subjected to an alkaline solution (preferably NaOH) in combination with a detergent (preferably SDS). In this environment, the cell wall structures are destroyed thereby releasing the polynucleotide of interest and other cell related compounds. Finally, the solution is neutralized by addition of a solution of an acidic salt, preferably an acetate, in particular potassium acetate (KAc) or sodium acetate (NaAc). The alkaline conditions lead to denaturation of pDNA by unwinding the supercoiled structure. Up to a pH-value of 12 to 12.5 the complete separation of the complementary strands is prevented. This enables entire renaturation of the plasmid molecule, when the pH is decreased again. If the pH-value exceeds the renaturation limit, the unseparated regions are lost and the pDNA is irreversibly denatured. At this stage the polynucleotide contains large domains of single stranded material (with a large exposure of hydrophobic bases) (Diogo M M, Queiroz J A, Monteiro G A, Prazeres D M F (1999) Analytical Biochemistry 275:122). The exact pH-value for irreversible denaturation of the plasmid is strongly influenced by the base pair composition, the resulting hydrogen bonds and its size (WO 97/29190). In parallel, genomic DNA and proteins are denatured, too. Denaturation of DNA leads to entanglement and formation of long single pair strands with low mechanical stability. Impact of mechanical stress may cause breakage of DNA, especially of the large gDNA molecules. The resulting fragments have properties comparable to those of pDNA. Since precipitation during the subsequent neutralization step is a size dependent process, these fragments may remain soluble and thus behave similarly to pDNA (Marquet M, Horn N A, Meek J A (1995) BioPharm September:26). Therefore they would interfere during the isolation process. The incubation time at high pH value is critical for the renaturation of the target polynucleotide, the degree of cell disintegration and the genomic DNA content in the preparation. Therefore the main parameter for quality and quantity of the polynucleotide preparation is the contact time with the alkaline lysis solution. Usually RNAse is added to the suspension to digest RNA into small pieces not to interfere the isolation process (Sambrook J, Fritsch E F, Maniatis T, (1989) Molecular Cloning: A Laboratory Handbook, CSH Press, Cold Spring Harbor, USA). After addition of NaOH and SDS, the solution becomes highly viscous. Local pH extremes, which irreversibly denature the plasmid (Rush M G, Warner R C (1970) J Biol Chem 245:2704) have to be avoided. Fast and efficient mixing has to be guaranteed in order to achieve a homogenous solution. Usually small containers like glass bottles containing the viscous solution are mixed very gently by hand (QIAGEN® Plasmid-Handbuch January 2001, Qiagen GmbH, Germany). This procedure can only be performed in a batchwise mode with a maximum of about 5 l lysate per bottle. It is mainly operator dependent, providing low reproducibility and is therefore not suited for a manufacturing scale. For large scale conventional stirrers are not suited because they may cause damage to pDNA and gDNA. Some processes use optimized tanks and stirrers or a combination of different mixers in order to overcome these problems (Prazeres D M F, Ferreira G N M, Monteiro G A, Cooney C L, Cabral JMS (1999) Trends Biotechnol 17:169; WO 02/26966).
In the subsequent neutralization step, cell debris, proteins as well as genomic DNA are co-precipitated with SDS by formation of a complex floccose precipitate (Levy M S, Collins I J, Yim S S, et al. (1999) Bioprocess Eng 20:7). Again gentle, but homogeneous blending (homogeneous neutralization) is essential for complete precipitation and for maintenance of pDNA quality. Vigorous mixing causes destruction of the plasmid and the flocks, resulting in redissolution of the impurities precipitated before (Levy M S, Ciccolini L A S, Yim S S, et al. (1999) Chem Eng Sci 54:3171; Marquet M, Horn N A, Meek J A (1995) BioPharm (September):26). This burdens the subsequent chromatographic separations (by e.g. loss of capacity for pDNA or the negative impact on the separation of RNA and gDNA, which have similar binding properties).
In the next step that follows alkaline lysis and neutralization, the precipitate has to be separated from the plasmid containing solution (this step is, in the meaning of the present invention, termed “clarification step”). In view of further purification by means of a resin, it is often necessary to adjust the parameters of the solution (like salt composition, conductivity, pH-value) to guarantee binding of the desired polynucleotide on the resin (this step is, in the meaning of the present invention, termed “conditioning step”). Subsequently, the solution is subjected to the first chromatographic step (capture step).
Centrifugation on fixed angle rotors (is the most frequently used method employed as the clarification step on laboratory and pre-preparative scales (Ferreira G N M, Cabral J M S, Prazeres D M F (1999) Biotechnol Prog 15:725). For lysate amounts usually handled in bottles the clear liquid phase separating from floating flocks and descending precipitate is sucked off and filtered. Otherwise the big flock-volume would shortly block the used filter. Since the fluid between the flocks contains residual plasmid DNA (Theodossiou I, Collins I C, Ward J M, Thomas O R T, Dunnhill P (1997) Bioprocess Engineering 16:175), high losses have to be taken into account. As further problem strong adsorption of nucleotides and pDNA to many filter-media has to be mentioned (Theodossiou I, Collins I J, Ward J M, Thomas O R T, Dunnhill P (1997) Bioprocess Eng 16:175; Theodossiou I, Thomas O R T, Dunnhill P (1999) Bioprocess Eng 20: 147). In many cases, bulk filter materials or bag filters are used for clarification of the lysate. Since these materials are either not certified or not scalable, they are not applicable for the production of pharmaceutical-grade plasmids on a manufacturing scale. More recent technologies utilize expanded bed adsorption (EBA), which allows removal of precipitated material while capturing the desired product (Chase H A (1994) Trends Biotechnol 12: 296). For capturing plasmid DNA direct after lysis by this chromatographic technique, it has to be taken into account that due to the large diameter of the (during neutralization built) aggregates of flocks pre-clarification prior to EBA is essential (Ferreira G N M, Cabral J M S, Prazeres D M F (2000) Bioseparation 9:1; Varley D L, Hitchkock A G, Weiss A M E, et al. (1998) Bioseparation 8:209).
There have been several attempts to develop improved technologies for each of the above-described steps. These attempts were mostly based on the following considerations:
Resuspension of the cells has to be carried out as fast as possible (especially when the cells have been frozen before), while avoiding high shear forces. Several commercially available types of stirrers are available for mixing the cell paste with the resuspension buffer in a batchwise mode in a vessel until homogeneity is achieved, the most commonly used device being a magnetic or impeller stirrer. Another method is described in US 2001 0034435 A1. Here the cell paste is diluted with a resuspension buffer and the cell/buffer mixture is circulated through a static mixer in a pump-around mode. It has also been suggested to directly dilute the fermentation broth with the resuspension buffer in a static mixer prior to lysis (WO 97/23601 A1).
For disintegration (lysis) of the cells in view of obtaining polynucleotides, several different methods have been suggested, e.g. methods that use thermal or chemical treatment. For the thermal lysis, a process using a flow-through heat exchanger (70-100° C.), in which the cells are continuously disintegrated after incubation of the resuspended cells in presence of a detergent and optionally lysozyme, is described (WO 96/02658 A1). Another physical method, which works in a temperature range of 70-90° C., is shown in WO 02/057446 A2:In a first step, the harvested cells are filtered utilizing filter aids and the resulting mixture is thermally lysed in a second step. Alternatively, disintegration can be carried out by pumping hot lysis buffer through the filter cake or by a flow through heat exchanger. Chemical lysis methods are operated at an alkaline pH-value, they are therefore referred to as “alkaline lysis”. A commonly used composition of the intrinsic lysis solution is described by Birnboim and Doly, but there are exist many variants of this solution. As the detergent that is part of lysis solution usually SDS is used, but other (e.g. non-ionic) detergents like TWEEN® or TRITON® are also suitable (e.g. WO 95/21250 A2). According to EP 0376080 A1, SDS is replaced by desoxycholate (DOC), while the three phase extraction method of U.S. Pat. No. 5,637,687 uses a novel composition for the cell-solubilization (benzyl alcohol+sodium iodide+guanidinium thiocyanate and/or guanidinium chloride). Most methods for alkaline lysis are operated in a batchwise mode. By way of example, the alkaline treatment can be carried out directly by adding a NaOH/SDS solution to a bacterial cell culture during exponential growth (in this case, no harvest of the cells is performed) or after resuspension of the cells in a proper buffer. Thereby, an alkaline solution is added until a pH value is reached that is 0.2 units lower than the pH value at which the pDNA-molecules are completely denatured, a pH value that is empirically determined and different for each single plasmid (WO 97/29190 A1). Another method utilizes a column comprising a carrier on a membrane filter that is capable of retaining a solution and permeating it by aspiration. When adsorbed onto the carrier, a certain amount of cells can be lysed in this column by means of lysozyme and further processed (EP 0814156 A2). A similar device that consists of a hollow body (tube) with a built-in filtration-layer is disclosed in EP 0616638 B1, EP 0875271 A2, and WO 93/11218 A1. Alkaline lysis is carried out in the part of the tube above the filtration section. The cell suspension and the used solutions are distributed and mixed in a non-continuous way.
The above-described methods are operated in non-continuous open systems that bear the risk of possible contamination. Handling and mixing is not automated and therefore user-dependent. The only way to handle larger pDNA-amounts, is multiplication of the devices, e.g. running them in parallel. These methods and devices are not suitable for production of pharmaceutical grade polynucleotides on a manufacturing scale. To achieve contacting and mixing of the cells with the lysis solution, it has also be suggested to use static mixers or simple tubings. This approach has been described for a cell lysis method, which is based on simply connecting the streams containing the pumped cell suspension and the lysing agent at a defined meeting point. The contact time is defined by the tubing volume (diameter and length of the tube) behind the meeting point and by the pump-velocity of the connected streams through the tubing. To facilitate rapid homogenization, the inner diameter of the tubing has to be reduced (2-8 mm) (WO 99/37750 A1). For connecting the two pumped streams at the meeting point, “Y”-connectors are proposed (WO 00/09680 A1). To enhance homogenous mixing of the cells with the lysis solution, especially designed static mixers are suggested. These devices are commercially available continuous flow-through supports. The contact time of the cells with the lysis solution is defined by the mixer dimensions and the flux (WO 97/23601 A1, WO 00/05358 A1). These online-contacting devices can also be combined with a subsequent stirred tank reactor. In this stirred tank reactor the neutralization step may also take place. (WO 02/26966 A2). Another process describes the combination of a static mixer, a so called “lysis coil” and an impeller (US 2001/0034435 A1).
The above-described continuous methods either work with simple connections of the flow stream or, in the case of using static mixers, with various fixtures, (e.g. helical structures.
Among the above-described methods, those using a simple tubing do not guarantee homogenous mixing, while the variant with the reduced tubing diameter (<1 cm) was designed for small-scale applications. The methods using static mixers (or reduced tubing diameters) may cause high shear forces to the polynucleotides.
In the neutralization step, normally an acidic solution containing potassium acetate is used. For concurrent precipitation of RNA, compositions that contain, in addition, sodium chloride, potassium chloride or ammonium acetate (up to 7 M) have been suggested (US 2001/0034435 A1). It was also shown that a solution containing divalent alkaline earth metal ions like CaCl2 that is added to the mixture after neutralization results in the precipitation of RNA and chromosomal DNA (U.S. Pat. No. 6,410,274 B1).
Neutralization of the lysed cell solution is often carried out as one single step in a batch mode. In EP 0814156 A2, WO 93/11218 A1, EP 0616638 B1, and EP 0875271 A2 the lysed cell solution is contacted with the neutralization/precipitation solution in the same device (column or tube with an in-line filter-material) like used before for the lysis step (already described above). Again, these techniques would be subject to several major limitations when transferred to the manufacturing scale production of pharmaceutical-grade polynucleotides, the problems also being possible contamination due to the non-continuous open system, user-dependence, and lacking scalability.
For the neutralization step, a stirred tank reactor that already contains the lysed cell solution, has been suggested, into which the neutralization solution is filled under continuous mixing with the stirrer at a speed of 500 rpm (WO 02/26966). A similar method is claimed in US 2001/0034435 A1, according to which neutralization is achieved by mixing the solutions with an impeller in a chilled jacketed holding tank or before in an in-line static mixer. Two very simple continuous contacting techniques are disclosed in WO 99/37750 A1 and WO 00/09680 A1. Both methods use the same setup as already described above for the lysis step connecting the two pumped streams at a meeting point with a reduced inner diameter of the resulting tubing (WO 99/37750) or a simple “Y” connector and tubing (WO 00/09680). For both methods, static mixers may be used in the neutralization step (WO 97/23601 A1, WO 00/05358 A1). These mixers are utilized in the same manner already described above for the lysis step.
The contact time of the pDNA-with the lysis solution has a major impact on its quality and depends on the time point and effectiveness of the neutralization step. Therefore, mixing of the lysed cell solution with the neutralization solution has to be fast and homogenous. This requirement can not be met by the techniques utilizing stirred tank reactors. Fast mixing with an impeller may cause rupture of the precipitated flocks and re-dissolution of impurities. The methods using a simple tubing do not guarantee homogenous mixing, while the variant with the reduced tubing diameter (<1 cm) may also cause undesired destruction of the flocks and is not suitable for larger scales. Although static mixers are expected to achieve homogenous mixing, they may get blocked due to the large volume of the flocks. Another disadvantage is that genomic DNA may be sheared by the internal structure of the mixer to a size, which will cause problems in the subsequent purification steps, let alone the possible negative impact of the mechanical stress to the desired polynucleotide.
To obtain a cleared lysate; the precipitated material has to be separated from the polynucleotide containing solution. Conventionally this clarification step is carried out in a batchwise mode using techniques known in the art like filtration or centrifugation (e.g. US 2001/0034435 A1, WO 02/04027 A1). Most commonly, the filters are depth filters (WO 00/09680). Other filter means for macrofiltration are macroporous diaphragms consisting of e.g. compressed gauze or an equivalent filter material (EP 0376080 A1). According to some protocols, filtration is carried out in presence of a filter aid (WO 95/21250 A2, WO 02/057446 A2, US 2002/0012990 A1). WO 96/21729 A1 discloses a method that contains a filtration step using diatomaceous earth after a centrifugation step, thereby achieving the additional effect of reducing the RNA content. Furthermore, combinations of a membrane filter with a loose matrix (glass, silica-gel, anion exchange resin or diatomaceous earth), which concurrently act as carrier for DNA, have been described (EP0814156A2). According to WO 96/08500 A1, WO93/11218 A1, EP 0616638 B1 and EP 0875271 A2, clarification is achieved by a device that has been described above for the lysis and for the neutralization step, whose filtration part may consist of different materials (e.g. glass, silica-gel, aluminum oxide . . . ) in the form of loose particles, layers or filter plates (especially with an asymmetric pore size distribution). The flux through the filter is accomplished by gravitation, vacuum, pressure or centrifugation. As a continuous clarification method, centrifugation (e.g. disc stack centrifuge or decanting centrifuge) are mentioned (WO 99/37750 A1, WO 96/02658 A1). Also combinations of centrifugation followed by filtration are described for the clarification purpose (WO 02/26966 A2, WO 96/02658 A1).
The above-described clarification methods are usually carried after the material has been incubated with the neutralization buffer for a certain period of time. This does not allow continuous connection with the foregoing steps and is only suitable for the laboratory scale. Apart from this, filtration techniques are usually carried out in open devices with the risk of possible contamination. Since any material that is used in a cGMP process must be validated, additional filter aids that might improve performance of the filtering process, are usually avoided.
In general, conventional filters have a limited capacity and are soon blocked by the large amount of voluminous flocks. In addition, a constant flux over the precipitate that is retained by the material may result in destruction of the flocks and re-dissolution of impurities, which would again have a negative impact on the following steps. For larger amounts of pDNA it has been suggested for some devices to multiply them (e.g. run them in parallel), which is insufficient for operating on a manufacturing scale. Centrifugation could be applicable continuously, but due to the sensitivity of polynucleotides to shear forces this treatment may also cause degradation of plasmid DNA and genomic DNA and detachment of precipitated impurities by rupture of the flocks.
In the subsequent conditioning step, the salt composition and/or the conductivity and/or the pH-value of the cleared lysate is adjusted to a value (to be determined empirically) that ensures binding to the resin in the subsequent capture step. Several conditioning methods have been described, e.g. in WO 97/29190 A1, WO 02/04027 A1 and WO 98/11208 A1. In the methods described in EP0814156A2, WO93/11218 A1, EP 0616638 B1 and EP 0875271 A2 the conditioning step is carried out as a washing and eluting step in the same device in which the previous steps took place.
Furthermore, as a pretreatment before the final purification, addition of an “Endotoxin Removal (ER) Buffer” (QIAGEN®) (WO 00/09680 A1) or TRITON X®-114 (WO 99/63076 A1) has been suggested.
Common to all of the described methods is their non-continuous and non-automated mode of operation that does not connect the operational steps.
For capturing the polynucleotide of interest, several techniques are known in the art, e.g. tangential flow filtration (WO 01/07599 A1), size exclusion chromatography (WO 96/21729 A1, WO 98/11208), anion exchange chromatography (WO 00/09680 A1, U.S. Pat. No. 6,410,274 B1, WO 99/16869), hydrophobic interaction chromatography (WO 02/04027 A1).
It has already been suggested combining some of the steps described above, e.g. for the processes described in EP 0814156 A2, WO 93/11218 A1, EP 0616638 B1 and EP 0875271, according to which cell lysis, neutralization, clarification, washing, optionally conditioning and capturing are carried out in the same apparatus. Typically, these methods are open systems that are operated in a non-automated/non-continuous mode including several holding steps. The devices are only suitable for the laboratory scale and cannot be transferred into manufacturing scale. The techniques also lack of reproducibility and suitability for cGMP large-scale production.
Alternatively, combinations utilizing different devices have been described, in which the individual steps are directly connected with each other.
The continuous combination of two ore more steps has been described in several patent documents: WO 96/02658 A1 describes the combination of thermal lysis and clarification by means of a centrifuge, WO 00/09680 A1 and WO 02/26966 A2 suggest combining alkaline lysis and neutralization. The methods described in US 2001/0034435 A1 and WO 97/23601 A1 combine the three steps resuspension of the cells, alkaline lysis and neutralization; WO 00/05358 A1 and WO 99/37750 A1 describe the combination of alkaline lysis, neutralization and clarification by centrifugation.
None of these processes combines more than three steps of the isolation procedure, the first step being the resuspension step and last one being the capture step. The devices used in these methods for contacting the solutions during lysis and neutralization do either not guarantee homogenous mixing or may apply disadvantageous shear forces to the solutes.